The physiology of hepatic ureagenesis has posed one of the more complex problems in metabolic regulation. this proposal entails a comprehensive evaluation of the mechanism(s) regulating glutamine metabolism and urea production in isolated hepatocytes obtained from acute or chronically acidotic or alkalotic rats. The primary questions to be addressed are: 1) What is the role of TCA-cycle metabolism in the control of hepatic ureagenesis? 2) How do changes in HCO3 and/or PCO2 alter urea formation from glutamine? 3) How do changes in K+, Pi and Ca2+ homeostasis alter ureagenesis and what is the control site? 4) Do hormones (insulin, glucagon, catecholamine) enhance or inhibit changes in urea formation which occur in response to altered acid-base homeostasis? 5) What is the site(s) of the hormonal action? Is it linked to alterations of intracellular pH, Ca2+, Pi or to changes of phosphatidylinositol turnover, activation of protein kinase C and/or modulation of the Na+-H+- exchanger? Hypotheses to be explored include: (a) that the initial signal in evoking pH modulation of urea formation from glutamine is an alteration of TCA-cycle metabolism; (b) that in addition to H+- homeostasis changes in intracellular Ca2+ or Ca2+/Pi ratio modulate urea formation secondary to alteration of TCA-cycle metabolism; (c) that the hormonal regulation of ureagenesis is mediated through changes in intracellular Ca2+ and thereby, modulation of TCA-cycle metabolism and/or protein kinase C activity; (d) alternatively, hormones affect urea formation from glutamine secondary to their effect on the Na+-H+- exchanger and hence, changes in intracellular pH, and fluxes via the glutaminase and glutamate dehydrogenase pathways, and therefore, ammonia availability for urea synthesis. We will explore these themes by incubating isolated hepatocytes with 15N and/or 13C labeled substrates. Studies will be carried out during induction of acute acidosis and alkalosis or following induction of chronic acidosis and alkalosis in the presence and absence of metabolic modulators. We will define precursor-product relationships and flux rates using GC-MS and/or NMR. The proposed experiments are of scientific as well as clinical importance by deepening our understanding of hepatic glutamine metabolism and urea synthesis in response to perturbations of H+ associated with/or independent of other physiological factors. The results we obtain should provide a panoramic view of the mechanism(s) regulating hepatic ureagenesis.